Methods and compositions for the treatment of asthma

ABSTRACT

The present invention provides methods of treating an inflammatory disorder, or a disorder having an inflammatory component, in a mammalian subject in need of such treatment by administering to said subject an effective amount of a composition in unit dosage form for delivery of a daily dose of said composition, said composition consisting essentially of: (i) an effective amount of γ-linolenic acid (GLA) for increasing dihomogammalinolenic acid (DGLA) levels in the inflammatory cells of said mammalian subject, thereby inhibiting the metabolism of arachidonic acid; (ii) an effective amount of a Δ 5  desaturase inhibitor for inhibiting accumulation of arachidonic acid in the serum of said mammalian subject; and, optionally, (iii) an effective amount of a competitive inhibitor of arachidonic acid metabolism. Preferred formulations may be in the form of a good tasting, preferably milk or fruit based drink, or a dried powder. The compositions reduce inflammation and inhibit the increase in serum arachidonic acid associated with gamma-linolenic acid.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. Ser. No. 09/644,380,filed Aug. 23, 2000 now U.S. Pat. No. 7,138,431 (allowed), which is acontinuation-in-part of PCT/US99/03120, filed Feb. 12, 1999, which is acontinuation-in-part of U.S. Ser. No. 09/028,256, filed Feb. 23, 1998(now U.S. Pat. No. 6,107,334); the disclosures of which are incorporatedherein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates generally to the fields of lipidmetabolism and dietary supplementation. More particularly, it concernscompositions and methods for controlling or reducing symptoms ofinflammation or inflammatory conditions that include the use ofunsaturated fatty acids, unsaturated fatty acid precursors, and/orunsaturated fatty acid analogs in nutritional supplements.

DESCRIPTION OF RELATED ART

Arachidonic acid (AA) is a polyunsaturated fatty acid found inrelatively small quantities in membranes of mammalian cells. Researchover the last four decades has shown that the in vivo modulation oflevels of arachidonic acid and oxygen-containing derivatives ofarachidonic acid (known as eicosanoids) is intimately liked to humandisease (for a review, see Samuelsson et al., Science, 237:1171-1176,1987 and Chilton et al, In: Crystal, West and Barnes, eds., Lung:Scientific Foundations, Lippincott-Raven Publishers, Chapter 6, 77-88,1997). For example, during inflammation, low levels of certainarachidonic acid derivatives render a protective response leading toenhanced disease resistance. However, these same molecules induce anautotoxic response leading to a variety of inflammatory disorders whenproduced in excessive quantities. Over the past three decades, thetherapeutic utility of blocking the metabolism of arachidonic acidthrough multiple pathways including 5-lipoxygenase and cyclooxygenase Iand II has become evident for the treatment of a wide range ofinflammatory disorders.

Since arachidonic acid or its precursors found in cells and tissues mustbe derived from diets, it follows that diet may affect diseasescontrolled by arachidonic acid or its derivatives. This relationship wassuggested in the 1960s by studies which showed differences infrequencies of inflammatory disorders among Greenland Eskimos and Danes(Chilton et al., Biochim. Biophys. Acta, 1299:1-15, 1996; Dyerberg andBang, Lancet, ii:443435, 1979). Later studies showed similar differencesbetween Japanese and Americans. These differences (Danes and Americanshave much higher frequencies of inflammatory disorders including asthma,arthritis, psoriasis and acute myocardial infarction) were attributed,in large part, to the consumption by Danes and Americans, on Westerndiets, of high dietary quantities of precursor fatty acids ofarachidonic acid (termed n-6 fatty acids) and arachidonic acid, offsetby the low consumption of n-3 fatty acids.

Based on these observations, a number of dietary fatty acid reductionand supplementation strategies were undertaken in an attempt toinfluence arachidonic acid metabolism, eicosanoid production andclinical outcomes. These studies carried out over the last two decadeshave revealed that controlling dietary fatty acid intake in a number ofanimal models has great potential in reducing eicosanoid synthesis andameliorating inflammation in models which mimic human arthritis, asthma,or glomerulonephritis (Prickett et al., J. Clin. Invest., 68:556-559,1981; Kelley et al., J. Immunol., 134:1914-1919, 1985; Lefkowith et al.,J. Immunol., 145:1523-1529, 1990; Rovin et al., J. Immunol.,145:1238-1245, 1990; Hurd et al., J. Clin. Invest., 67:476-482, 1981).

These studies demonstrated that the formation of derivatives of AA andthe subsequent effects of these compounds (eicosanoids) on cells andtissues are central processes in inflammation and allergy. Dietary fattyacid reduction and supplementation strategies have been utilized inanimals and humans in an attempt to modulate cellular AA levels andmetabolism, and to ameliorate clinical inflammatory disorders. However,dietary modifications in humans on Western diets have shown only modestefficacy. If these observations are to prove useful in the treatment ofsuch disorders, it is necessary to find more efficient dietarystrategies to reduce eicosanoid generation in humans and to determinethe mechanism(s) leading to this reduction.

In terms of inflammation, at least four dietary reduction andsupplementation strategies have been utilized in both animals and humansin an attempt to influence eicosanoid production and clinical outcomes.One strategy has been to supplement “normal” diets with n-3 fatty acids.Here, there has been some controversy as to how effective these fattyacids are in reducing lipid mediators (eicosanoids) of inflammation(Chilton et al., J. Clin. Invest., 91:115-122, 1993; Sperling et al., J.Immunol., 139:41864191, 1987; Strasser et al., Proc. Natl. Acad. Sci.USA, 82:1540-1543, 1984; Kojima et al., Dermatologica, 182:225-230,1991; Galloway et al., Clin. Sci., 68:449454, 1985; Mori et al, Lipids,22:744-750, 1987; Ahmed and Holub, Lipids, 19:617-724, 1984; Payan etal., J. Clin. Immunol., 6:402-410, 1986; Rosenthal and Hills, Biochem.Biophys. Acta, 875:382-391, 1986; Triggiani et al., J. Immunol.,145:2241-2248, 1990). For example, several studies report only modestinhibition of leukotrienes and PAF after n-3 fatty acid supplementation,while other investigations report more dramatic reductions (Chilton etal., 1993; Sperling et al., 1987; Strasser et al., 1984). The basis forthese discrepancies is unclear at this time. In addition to eicosanoids,n-3 fatty acids have been shown to affect processes such as geneexpression, cytokine generation and programmed cell death in a number ofin vitro and in vivo settings (Endres et al., N. Engl. J Med.,320:265-271, 1989; Clarke and Jump, Lipids, 31:87-S 11, 1996;Chandrasekar et al., J. Autoimmunity, 8:381-393, 1995; Fernandes et al.,J. Immunol., 152:5979-5987, 1994).

A second strategy to effect changes in AA metabolism in humans has beento remove dietary essential fatty acids from the diet. This eliminatessources of cellular AA that are derived from dietary linoleic acid (LA).Severe restrictions of LA intake in infants result in significant fallsin levels of prostaglandin metabolites (Friedman et al., Pediat. Res.,12:711-714, 1978). Wene and colleagues studied healthy men on fat-freeeucaloric diets and found that LA levels in serum components felldramatically within seven days of starting the diet (Wene et al., J.Clin. Invest., 56:127-34, 1975). However, if calorie intake was thenreduced (intermittent fasting), LA levels in serum increased. This LArepletion may be due to mobilization of fatty acids from adipose tissuetriglycerides.

A third strategy to reduce AA metabolism has been to restrict preformedAA in diets of humans. There are several conflicting studies in humansrestricting preformed AA by the chronic avoidance of animal tissue withresults varying from increases to moderate reductions in serum AA levels(Phinney et al., Am. J. Clin. Nutr., 51:385-392, 1990; Sanders et al,Am. J. Clin. Nutr., 31:805-813, 1978; Melchert et al., Atheroscler.,65:159-166, 1987). In contrast to studies restricting dietary AA, humanssupplemented with AA (an additional 6 g/day) exhibit a pronouncedincrease in AA levels within plasma triglycerides, phospholipids,cholesterol esters, and platelet phospholipids (Seyberth et al., Clin.Pharmacol. Ther., 18:521-529, 1975). This increase within complex lipidsis accompanied by an increase in eicosanoid generation and a markeddecrease in the ADP threshold dose required to induce plateletaggregation.

A fourth strategy that has been utilized to influence AA metabolism isto supplement normal diets with oils (primrose and borage) rich in gammalinolenic acid (18:3, n-6). Such oils have been shown to improveclinical symptoms of patients with atopic dermatitis and rheumatoidarthritis (Leventhal et al., Ann. Intern. Med., 119:867-873, 1993;Miller et al., J. Nutr., 120:36-44, 1990; Horrobin, J. Lipid Res.,31:163-194, 1992; Zibok and Fletcher, Am J. Clin. Nutr., 55:39-45, 1992;Tate et al., J. Rheum., 16:729-734, 1989). The mechanisms by which GLAinfluences these inflammatory disorders has not been elucidated. Infact, it is paradoxical that providing a dietary precursor of AA, GLA,attenuates inflammation. It is known that a portion of the GLA providedis elongated (by 2 carbons) in vivo to form dihomogammalinolenic acid(DGLA) (Horrobin, 1992; Zibok and Fletcher, 1992; Tate et al., 1989).DGLA can then be metabolized to oxygenated Products, 15-OH-20:3. (15HETrE) and prostaglandin E, by 15 lipoxygenase and cyclooxygenase,respectively (Miller et al., 1990; Horrobin, 1992). PGE₁ has been foundto be anti-inflammatory in a variety of in vitro systems and animalmodels (Kerins et al., Prog. Hemostasis Thromb., 10:307-337, 1991). GLAsupplementation also has been shown to reduce the capacity of some cellsto produce AA-derived eicosanoids (Leventhal et al., 1993; Zibok andFletcher, 1992).

The inventor's laboratory has provided humans on controlled diets with awide range of dietary fatty acid supplements and supplement combinationsin an attempt to affect AA metabolism in humans (Chilton et al., 1993;Triggiani et al., 1990; Chilton-Lopez et al., J. Immunol.,156:2941-2947, 1996; Johnson et al., J. Nutr., 127:1435-1444, 1997). Inthese studies, well defined diets (prepared and fed in a GeneralClinical Research Center [GCRC]) and measurement techniques (negativeion chemical ionization GC/MS) that precisely determine fatty acid andeicosanoid levels in serum and inflammatory cells have been utilized.

Although much work has been performed on the dietary supplementation offats, many questions remain to be answered, including the determinationof the capacity of different inflammatory cells to synthesize (elongateand desaturate) polyunsaturated fatty acids; the major mechanism(s) bywhich analogs (which can be induced by dietary supplementation) of AAinfluence eicosanoid generation and the development of dietarystrategies that will produce natural antagonists of AA in inflammatorycells, thereby reducing the synthesis of pro-inflammatory eicosanoidswithout increasing serum levels of AA. These and other questions are atleast partially addressed by the present disclosure.

SUMMARY OF THE INVENTION

The present invention is directed to dietary strategies that treat, orreduce the side effects of inflammatory disorders such as asthma andarthritis. Although GLA has been reported as beneficial in reducingsymptoms of certain inflammatory conditions, unfortunately, dietarysupplementation with GLA results in an increase in serum arachidonicacid (AA), with potentially undesirable effects. In studies disclosedherein, however, it is shown that GLA supplementation does not increaseAA in certain inflammatory cells. Also as disclosed herein, neutrophils,the inflammatory response cells, do not possess a Δ⁵ desaturaseactivity, as do hepatocytes. Thus, the product of GLA elongation, DGLAcannot be converted to AA and eicosanoids in inflammatory cells. Inserum, however, DGLA formed from the elongation of GLA is converted toAA via the action of a Δ⁵ desaturase. This build-up of serum AA islikely to have harmful consequences in humans. For example, previousstudies have demonstrated that increases in AA of this magnitude canincrease platelet reactivity, which is undesirable in most cases.

As disclosed herein, these potentially harmful effects can becircumvented by providing a Δ⁵ desaturase inhibitor in combination withthe GLA, thus preventing the increase in serum AA levels upon GLAadministration. In addition, stearidonic acid or ω-3 arachidonic acidmay be provided as antagonists of arachidonic acid metabolism in immunecells, because, as shown herein, stearidonic acid is taken Up by humanneutrophils and elongated to ω-3 arachidonic acid, also a competitiveinhibitor of arachidonic acid metabolism. It is contemplated that abuildup of ω-3 arachidonic acid in neutrophils may also result infurther inhibition of the serum Δ⁵ desaturation of DGLA in hepatocytes,resulting in further inhibition of serum arachidonic acid accumulation.

Described herein are compositions for diminishing symptoms ofinflammatory disorders. The compositions include γ-linolenic acid ordihomogammalinolenic acid, Δ⁵ desaturase inhibitors, and ω-3 competitiveinhibitors of arachidonic acid metabolism. In preferred embodiments thedescribed ingredients include from around 80% to about 95% purepolyunsaturated fatty acids. Preferred Δ⁵ desaturase inhibitors includeeicosapentaenoic acid, sesamin, episesamin, ses aminol, sesamolin,curcumin, α-linolenic acid, heneicosapentaenoic acid, docosahexaenoicacid, alkyl gallate, propyl gallate, and p-isopentoxyaniline. Theseinhibitors may be provided as free fatty acids, fatty acyl esters,diglycerides, triglycerides, ethyl esters, phospholipids, steryl esters,sphingolipids, or a combination of these. In certain embodiments, acompetitive inhibitor of arachidonic acid metabolism may be ω-3arachidonic acid or stearidonic acid. In certain embodiments acompetitive inhibitor of inflammatory cell AA metabolism and liver Δ⁵desaturase may be ω-3 AA or stearidonic acid (SA).

Preferred formulations of the disclosed compositions include flavoredliquids or powders that may be rehydrated to form a drink. Preferredformulations may also include ingredients such as water, corn syrup,maltodextrin, sodium caseinate, calcium caseinate, soy protein,magnesium chloride, potassium citrate, calcium phosphate tribasic, orsoy lecithin. The disclosed formulations may also include variousemulsifying or stabilizing agents and antioxidants known in the art.Suitable emulsifying or stabilizing agents include, without limitation,xanthan gum, guar gum, pectin, carob seed gum (locust-bean gum),tragacanth gum, methylcellulose, alginates, carrageenan or the like.Additional preferred ingredients may include sucrose, glucose,aspartame, glycerol, sorbitol, sorbic acid, galactolipids,sphingolipids, lecithins, cellulose, hydroxypropylmethylcellulose, maltor malt extract, gelatin, casein, cholesterol, egg yolk, sodium dodecylsulfate, benzalkonium chloride, p-hydroxybenzoic acid, vitamin C,vitamin E or alpha-tocopherol. A composition in the form of a driedpowder may be prepared using any suitable pharmaceutical carrier(s)routinely used for preparing solid formulations. Examples of suchcarriers include magnesium stearate, starch, lactose, sucrose andcellulose.

In certain embodiments, the disclosed compositions are contained in anessentially oxygen-free, air-tight container. By oxygen-free is meantthe ambient air trapped within the container is essentially free ofoxygen as is achieved, for example, by sealing the container in anoxidatively inert gas environment, such as a nitrogen gas environment.Preferred containers include cans or foil pouches that provide apunch-through opening for a straw. The compositions may also include aflavoring agent such as a fruit flavoring agent or a fruit juice. Otherflavoring agents may include vanilla, chocolate, eggnog, berry, or otherflavoring agents known in the art. Preferred antioxidants includebeta-carotene, vitamin E, vitamin C, selenium, alpha tocopherol, andtaurine.

Certain compositions disclosed herein may be described as milk baseddrinks for treatment of inflammatory disorders that may include anunsaturated fatty acid portion containing γ-linolenic acid ordihomogammalinolenic acid, a Δ⁵ desaturase inhibitor, and stearidonicacid or ω-3 arachidonic acid. Alternatively, certain compositions,disclosed for treatment of an inflammatory disorder, may includeγ-linolenic acid or dihomogammalinolenic acid, eicosapentaenoic acid,and stearidonic acid or ω-3 arachidonic acid. Such formulations may beused in the treatment of asthma, allergic rhinitis, allergicrhinoconjunctivitis, psoriasis, acute myocardial infarction,glomerulonephritis, Crohn's disease, inflammatory bowel disease, orarthritis, for example. The compositions are also effective fortreatment of conditions that have an inflammatory component thatincludes a role for arachidonic acid metabolites, such as, for example,breast cancer, colon cancer, prostate cancer, autoimmune diseases, e.g.systemic Lupus erythematosus, schizophrenia, depression, IgAnephropathy, sepsis and toxic shock, organ failure, organ transplants,coronary angioplasty, risk reduction for Alzheimer's disease, cysticfibrosis, atherosclerosis, menstrual discomfort, cyclic breast pain,premature labor, gout, venous leg ulcers, chronic urticaria, primarydysmenorrhea, endometriosis, and Lyme disease.

The compositions disclosed herein, including milk-based liquids havingan unsaturated fatty acid portion, may contain from about 80-95% pureγ-linolenic acid, eicosapentaenoic acid, and stearidonic acid. Theseunsaturated fatty acids may be isolated from natural sources such asplants or animal tissues, or they may be isolated from transgenic cellsengineered to produce at least one of the unsaturated fatty acids.Transgenic cells are defined as cells that include at least one stableheterologous gene, that, in this case are involved in producing thedesired polyunsaturated fatty acid. Such genes may encode enzymesinvolved in a pathway that converts a precursor into the desiredproduct, or that produce a precursor of the desired product, forexample. Transgenic cells may include animal cells, yeast cells, plantcells, bacterial cells, or cyanobacterial cells, for example. It is alsounderstood that such cells may be contained in an organism such as ananimal, a plant, or a plant organ.

In certain embodiments, the present disclosure provides a method ofinhibiting increases in serum arachidonic acid in a mammal to whichγ-linolenic acid (GLA) has been provided, comprising providing to themammal a Δ⁵ desaturase inhibitor. In particular aspects, the mammal hasan inflammatory disorder. In particularly preferred embodiments, the Δ⁵desaturase inhibitor is eicosapentacnoic acid (EPA). Other Δ⁵ desaturaseinhibitors contemplated to be useful in the present invention includesesamin, episesamin, sesaminol, sesamolin, curcumin, heneicosapentaenoicacid, alkyl gallate, propyl gallate, p-isopentoxyaniline, anddocosahexaenoic acid. In such embodiments, an ω-3 competitive inhibitorof inflammatory cell AA metabolism and liver Δ⁵ desaturase activity mayalso be provided. Preferred examples are stearidonic acid and ω-3arachidonic acid.

The GLA, EPA, and SA may be administered as free fatty acids or as fattyacyl esters. In particular aspects, the acyl esters may betriglycerides, ethyl esters, phospholipids, steryl esters orsphingolipids. The GLA, EPA, and SA may be administered in a singlepharmaceutical or nutritional composition or as distinct pharmaceuticalcompositions or nutritional supplements. Preferred compositions arecontained in a good tasting, milk based or juice based drink.

Particular aspects of the present invention provide a method of treatingan inflammatory disorder in a mammal comprising providing to the mammala γ-linolenic acid in an amount effective to increase the amount ofdihomo-γ-linolenic acid (DGLA) in inflammatory cells and the circulationof the mammal; a Δ⁵ desaturase inhibitor in an amount effective toinhibit the formation of arachidonic acid in the serum of the mammal;and an amount of stearidonic acid effective to inhibit arachidonic acidmetabolism in immune cells; wherein the increase in DGLA in theinflammatory cells of the mammal inhibits the metabolism of arachidonicacid and decreases the inflammatory response in the mammal. Theinflammatory diseases may include, for example, asthma, allergicrhinitis, allergic rhinoconjunctivitis, arthritis, psoriasis, acutemyocardial infarction, glomerulonephritis, Crohn's disease, inflammatorybowel disease, or any disease that is mediated by lipid inflammatorymediators as described herein.

Also contemplated herein is a dietary supplement preparation consistingessentially of GLA in an amount effective to increase the DGLA level inthe user, such that the DGLA inhibits the metabolism of arachidonic acidin the inflammatory cells, and an amount of EPA which is effective toinhibit accumulation of arachidonic acid in the serum of the user. Thedietary supplement of the invention is readily adapted foradministration in unit dosage form for convenient delivery of a dailydose that consists essentially of GLA, present in an amount of from atleast about 1 gram to about 15 grams, preferably about 1 gram to about10 grams and most preferably about 1.5 to about 3 grams; EPA, present inan amount from about 0.1 grams to about 10 grams, preferably about 0.25grams to about 5 grams and most preferably about 0.5 grams to about 3grams; and, optionally, stearidonic acid (SA), present in an amount fromabout 0.1 gram, or even 1 gram to about 15 grams, preferably about 2grams to about 10 grams and most preferably from about 3 grams to about5 grams. When operating below the ranges specified the desired effectson eicosanoid synthesis and prevention of arachidonate accumulation willnot be obtained. Operating above the indicated ranges will result in theconsumption of large quantities of oils and may result in undesirableeffects due to the large caloric intake from these oils.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that, the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Biochemical desaturation/elongation of essential fatty acids topolyunsaturated fatty acids.

FIG. 2. Dose-response of GLA supplementation on serum fatty acid levels.

FIG. 3. Effect of GLA supplementation (up to 12 weeks) on fatty acidlevels in human serum.

FIG. 4. Dose-response of GLA and metabolites supplementation intoneutrophil lipids.

FIG. 5. Effect of GLA supplementation (up to 12 weeks) on fatty acidlevels in human neutrophils.

FIG. 6A and FIG. 6B. Incorporation of AA (FIG. 6A) and DGLA (FIG. 6B)into glycerolipid classes of neutrophils.

FIG. 7. Fatty acid release from stimulated neutrophils before and aftersupplementation.

FIG. 8. Influence of GLA supplementation on leukotriene generation.

FIG. 9. Influence of GLA supplementation on 5-lipoxygenase activity.

FIG. 10. In vitro metabolism of GLA in human neutrophils.

FIG. 11. Metabolism of ¹⁴C-DGLA to products by stimulated neutrophils.

FIG. 12. Influence of 15-HETrE on leukotriene generation.

FIG. 13. In vitro metabolism of stearidonic acid in human neutrophils.

FIG. 14. In vitro metabolism of stearidonic acid in human neutrophils.

FIG. 15. Metabolism of GLA by human eosinophils.

FIG. 16. Influence of borage oil on early and late asthmatic response.

FIG. 17A and FIG. 17B. The two in vivo approaches to be used in order tosynthesize close structural analogues of AA without affectingcirculating AA levels. FIG. 17A. GLA supplementation in combination withEPA. FIG. 17B. Stearidonic Acid Supplementation.

FIG. 18A. Bar graph indicating inhibition of arachidonic acid synthesisin liver cells by Δ⁵ desaturase inhibitor, eicosapentaenoic acid.

FIG. 18B. Per cent inhibition of arachidonic acid synthesis in livercells by Δ⁵ desaturase inhibitor, eicosapentaenoic acid.

ABBREVIATIONS

-   AA, 20:4, arachidonic acid; EPA, 20:5 (n-3), eicosapentaenoic acid;    LA, 18:2, linoleic acid; EFA, essential fatty acid; PUFA,    polyunsaturated fatty acid; GLA, 18:3 (n-6), gammalinolenic acid;    DGLA, 20:3 (n-6), dihomogammalinolenic acid; SDA, 18:4 (n-3),    stearidonic acid; ω-3 AA, 20:4 (n-3); PC, phosphatidylcholine; PE    phosphatidylethanolamine; PL phosphatidylinositol; GPC,    sn-glycero-3-phosphocholine; GCRC, General Clinical Research Center;    GC/MS, gas chromatography/mass spectrometry; NICI negative ion    chemical ionization; TNF, tumor necrosis factor; FMLP,    n-formyl-methionine-leucine-phenylalanine; TLC, thin layer    chromatography; HPLC high pressure liquid chromatography; LTB₄,    leukotriene B₄; LTB₅, leukotriene B₅; LTC₄ leukotriene C₄; PAF,    platelet activating factor; HBSS, Hank's Balanced Salt Solution;    BALF, bronchoalveolar lavage fluid; EAR, early asthmatic response;    LAR, late asthmatic response.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present disclosure provides a dietary strategy, includingnutritional supplements, designed to improve or at least partiallyalleviate symptoms of inflammatory disorders by providing a combinationof polyunsaturated fatty acids, preferably in a milk or juice based,good tasting drink. The compositions and methods disclosed herein arosein part from the surprising discovery that human neutrophils lack a Δ⁵desaturase activity, and that, while the use of γ-linolenic acid (GLA)in the treatment of arthritis or other inflammatory conditions leads toan increase in arachidonic acid (AA) in serum phospholipids, thisincrease does not occur in neutrophils. An alternate and synergisticmethod of inhibiting neutrophil AA metabolism and preventing serumaccumulation of AA in response to increased GLA is also available inlight of the present discovery. It was contemplated that stearidonicacid (18:4) would also be elongated in neutrophils to form ω-3arachidonic acid, which would accumulate due to the lack of a Δ⁵desaturase activity (Δ⁵ desaturase produces AA from ω-3 arachidonicacid). This excess of ω-3 arachidonic acid is available, then, tocompete with natural AA (n-6) for enzymes (phospholipase A2 isotypes,cyclooxygenase isotypes, and 5-lipoxygenase) that convert AA tooxygenated metabolites. Concomitantly, ω-3 AA formed within the serummay be converted to eicosapentaenoic acid, possibly further inhibitingthe hepatic Δ⁵ desaturase, and thereby contributing to the inhibition ofaccumulation of serum AA.

The present disclosure, thus, represents in part, a defined, threepronged mechanism of decreasing symptoms of inflammatory disorders. Aprecursor of arachidonic acid, such as GLA, may administered to asubject in order to reduce inflammation, as in conventional treatments.GLA administration to humans has been shown to effectively block AAmetabolism, block the synthesis of AA products and mitigate the clinicalsymptoms of inflammatory disorders. As an additional element, theincrease in arachidonic acid that is normally seen in serum fatty acidswith administration of GLA may be inhibited by administering a Δ⁵desaturase inhibitor, such as eicosapentaenoic acid (EPA), for example.This combination can be utilized in humans to inhibit Δ⁵ desaturation ofDGLA to arachidonic acid in serum. Also disclosed herein is thesynergistic step of providing for the synthesis of close structuralanalogs (antagonists) of AA by providing stearidonic acid, a competitivesubstrate of inflammatory cell elongase activity, which in this case,leads to ω-3 arachidonic acid. Thus, the antagonist of AA metabolism inthe neutrophils and other inflammatory cells prevents the synthesis ofthe eicosanoids responsible for an inflammatory response without aconcomitant increase in serum AA.

The described strategy is based on the knowledge that when GLA isadministered as a dietary supplement, an endogenous elongase activity ininflammatory cells synthesizes a close analogue of AA, DGLA (FIG. 17A).A part of the present disclosure is that certain inflammatory cellscannot further desaturate DGLA to AA because they lack a Δ⁵ desaturase.However, in human circulation, GLA becomes elongated to DGLA, and thenis further desaturated to AA. This leads to a marked increase in AAlevel in the circulation as a result of GLA administration. Theincreased AA in the circulation has been shown to cause potentiallydetrimental effects such as increased platelet reactivity in humans(Seyberth et al., 1975).

The present invention includes a method of providing high concentrationsof GLA to humans without causing a concomitant accumulation of serum AA.Thus, high concentrations of GLA can be administered to humans tosynthesize DGLA in inflammatory cells, thereby inhibiting AA metabolism,eicosanoid synthesis and attenuating the signs and symptoms ofinflammatory disorders without the significant side effect ofcirculatory AA accumulation. Specifically in the present invention, GLAis administered to humans in combination with Δ⁵ desaturase inhibitorsincluding EPA. The present inventor has shown that this combination ofGLA and the Δ⁵ desaturase inhibitor, EPA, causes a marked accumulationof DGLA in the circulation and in inflammatory cell lipids withoutcausing an increase in accumulation of AA in serum lipids. Alsodescribed herein, the n-3 fatty acid, stearidonic acid (18:4) may beelongated in neutrophils to form ω-3 arachidonic acid (FIG. 1) resultingin a dose-dependent increase in ω-3 arachidonic acid in glycerolipids ofthese cells, and without an increase in the Δ⁵ desaturase product of ω-3arachidonic acid, eicosapentaenoic acid, nor an increase in AA. Thus,high levels of the AA analog, ω-3 AA, can be induced in inflammatorycells by providing inflammatory cells (in vitro or in vivo) withstearidonic acid, which may be converted to ω-3 AA to compete withnatural AA (n-6) for enzymes (phospholipase A₂ isotypes, cyclooxygenaseisotypes, and 5-lipoxygenase) that convert AA to oxygenated metabolites.

Thus, the present invention provides combined compositions of GLA, EPA,and optionally SA, for example, for the treatment of inflammatorydisorders such as psoriasis, rheumatoid arthritis, inflammatory boweldisease, Crohn's disease, ulcerative colitis, asthma, renalinflammation, atopic dermatitis, thyroiditis, or any other disease,syndrome, condition or disorder that is mediated by lipid inflammatorymediators. Included in the latter category are diseases such as breastcancer, colon cancer, prostate cancer, autoimmune diseases, e.g.systemic Lupus erythematosus, schizophrenia, depression, IgAnephropathy, sepsis and toxic shock, organ failure, organ transplants,coronary angioplasty, risk reduction for Alzheimer's disease, cysticfibrosis, atherosclerosis, menstrual discomfort, cyclic breast pain,premature labor, gout, venous leg ulcers, chronic urticaria, primarydysmenorrhea, endometriosis, and Lyme disease. To those skilled in theart it will be apparent that all of these conditions have aninflammatory component that includes a role for arachidonic acidmetabolites.

The present invention provides methods and compositions for altering theserum arachidonic acid levels of a mammal in need of GLA supplementationby providing a Δ⁵ desaturase inhibitor in an amount effective to preventor inhibit the accumulation of AA in the serum of said mammal. Inpreferred aspects, the present inventor has found that EPA is an in vivoand in vitro inhibitor of Δ⁵ desaturase activity in the liver of humans.Thus, administration of a combination of GLA and EPA will serve toprevent the synthesis of AA and its metabolites in neutrophils, whilstinhibiting the accumulation of AA in the serum. These methods andcompositions are discussed in further detail herein below.

Sources of Fatty Acids for use in Dietary Supplements

The fatty acyl compositions of the present invention may be obtainedfrom a variety of sources. These acids may form part of a phospholipid,steryl ester, a sphingolipid, a glyceride, such as a di- or triglycerideor may be present as free fatty acids. For a comprehensive treatise ofthe synthesis of fatty acyl containing lipids, one of skill in the artis referred to “Lipid: Chemistry, Biochemistry and Nutrition” (Mead etal, Lipid: Chemistry, Biochemistry and Nutrition, Plenum Press, NewYork, 1986). More particularly, the distribution of fatty acids intissue lipids is described in Chapter 5. Of particular relevance arechapters 11, 14, 15, 17, and 18 which describe synthesis and metabolicrelevance of eicosanoids, triacylglycerols, steryl esters,phosphoglycerides and sphingolipids.

GLA may be obtained from sources such as oils of evening primrose,borage, blackcurrant, and various fungi and algae including Mucor,Rhizopus and Spirulina. DGLA may be synthesized from GLA oralternatively, may be obtained from a variety of animal tissuesincluding, liver, kidneys, adrenals, or gonads. AA can also be isolatedfrom similar tissues, or from egg yolk, and can also be found in variousfungal and algal oils. EPA may be found in marine oils and various algaland fungal oils. Of course, although rather difficult and expensive, allthe fatty acids may also be chemically synthesized de novo.

It is also an aspect of the present disclosure that, because specific,purified fatty acids are desired, certain organisms may be engineered to“overproduce” these particular fatty acids, making them easier toisolate and purify. For example, bacterial cells, cyanobacterial cells,fungal cells, yeast cells, plant cells, animal cells, or even organs,organelles or whole plants or animals may be engineered to overproduceor even to secrete the fatty acids needed for the compositions disclosedherein.

For example, gene sequences may be isolated that encode a single enzymein the pathway leading to a fatty acid product, such as a Δ⁶ desaturasegene, for example, as described in U.S. Pat. No. 5,689,050,(incorporated herein by reference), for use in the practice of thepresent invention, or an entire pathway may be isolated from genomicclones, as described in U.S. Pat. No. 5,683,898 (incorporated herein byreference). In certain embodiments, an organism or a cell of an organismis selected that produces a precursor to a desired fatty acid, and insuch cases, genes encoding the “downstream” enzyme or enzymes may beprovided. It also understood that even if a cell produces the selectedfatty acid, the production may be enhanced or increased by supplyingadditional copies under the control of more active promoter regions, oreven inducible promoters so that expression of the genes may becontrolled. Such systems are well known in the art.

The present invention may be described in terms of methods of treatmentand pharmaceutical compositions, but it is understood that the GLA, EPA,SA and any other fatty acid used ill the practice of the presentinvention may be incorporated into a dietary margarine, milkshake, afraction of whole milk, a milk product, a juice, combination of juicesor fruit product or other foodstuff. Pharmaceutical and dietarycompositions comprising fatty acyl components are well known to those ofskill in the art and have been described in U.S. Pat. Nos. 4,666,701;4,576,758; 5,352,700; 5,328,691; 4,444,755; 4,386,072; 4,309,415;4,888,326; 4,965,075, and 5,178,873; in European Patent Nos. EP 0 713653, and EP 0 711 503; and in PCT Applications WO 96/31457 and WO97/21434 (each of which is specifically incorporated herein byreference).

Δ⁵ Desaturase Inhibitors

As discussed earlier, AA and compounds derived therefrom are centralmediators of inflammatory and allergic responses. A mechanism forameliorating the deleterious effects of these compounds is throughdietary control. One such manipulation involves the production or use ofnatural antagonists of AA at the sites of action of these compounds,inflammatory cells. Dietary supplementation with GLA has been shown tobe effective at lowering inflammatory response, and it appears thatalthough neutrophils (inflammatory response cells) take up GLA andelongate it to DGLA, there is no subsequent production of theeicosanoids that mediate inflammatory response. As shown herein, thiseffect occurs because neutrophils do not possess a Δ⁵ desaturase, thusthe DGLA produced is not desaturated to AA. However, althoughneutrophils lack a Δ⁵ desaturase, other cells in the circulatory systemdo have Δ⁵ desaturation capabilities and such cells readily elongate thesupplemented GLA to DGLA and desaturate that DGLA to AA. This increasedcirculatory AA is a potently harmful agent, and it is this problem thatis addressed as an aspect of the present disclosure. Based on thediscoveries disclosed herein, this potentially harmful accumulation ofAA in the circulation of GLA-supplemented individuals can now beprevented by a concomitant provision of a Δ⁵ desaturase inhibitor.

EPA is an ω-3, 20 carbon fatty acid that contains five double bonds(20:5), and as such is a structural analogue of AA (20:4). EPA has beenshown to act as a Δ⁵ desaturase inhibitor, presumably via a feedbackinhibition mechanism. Methods of producing this fatty acid have beenwell described in the art (e.g. U.S. Pat. Nos. 5,683,898; 5,567,732;5,401,646; 5,246,842; 5,246,841; 5,215,630 each incorporated herein byreference). The present invention, in preferred embodiments, employs EPAas a Δ⁵ desaturase inhibitor to be administered in a nutritionalsupplement to those individuals receiving GLA supplements, in order toprevent the accumulation of AA in the circulation of said individuals.

In certain embodiments, it is contemplated that other inhibitors of Δ⁵desaturase will also be useful, such compounds include members of thesesamin family, members of the curcumin family and other fatty acidssuch as docosahexaenoic acid, and heneicosapentaenoic acid. U.S. Pat.No. 5,674,853, which is specifically incorporated herein by reference,describes the use of lignins from the sesamin family in combination withsaponin compositions as enteral formulations for treatment of infectionand inflammation. Such sesamins will be useful in the context of Δ⁵desaturase inhibition as described herein.

U.S. Pat. No. 5,336,496, incorporated herein by reference, describesother inhibitors of Δ⁵ desaturase that may be useful in the context ofthe present invention. In general terms, the Δ⁵ desaturase inhibitorsdescribed therein include lignan compounds, curcumin and piperonylbutoxide. As used herein the term “lignan” includes compounds such assesamin, sesaminol, episesamin, episesaminol, sesamolin,2-(3,4-methylenedioxyphenyl)-6-(3-methoxy-4-hydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane;2,6-bis-(3-methoxy-4-hydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane; and2-(3,4-methylenedioxyphenyl)-6-(3-methoxy-4-hydroxyphenoxy)-3,7-dioxabicyclo[3.3.0]-octane.

Methods of producing and separating these compounds are well known tothose of skill in the art. For example U.S. Pat. No. 5,209,826 describesa method of separating sesamin and episesamin. It is contemplated thatthe present invention may use such methods in obtaining Δ⁵ desaturaseinhibitors. As such, U.S. Pat. No. 5,209,826 is incorporated herein byreference. In other embodiments, the present invention employsmicroorganisms or plants, for example, for producing fatty acids asinhibitors of Δ⁵ desaturase. Such techniques are well known to those ofskill in the art (e.g., Shimizu et al., 1988; Shimizu et al, 1989).

Methods for the synthesis of curcumin-related compounds have beendescribed in U.S. Pat. No. 5,679,864 (incorporated herein by reference).These methods involve reacting the enol form of a 2,4-diketone with amonocarbocyclic aldehyde in the presence of an organic amine catalyst.The reactants are dissolved in a highly polar, aprotic, organic solvent.The curcumin-related product is recovered in crystalline form byprecipitation from the reaction mass and solvent recrystallization andmay be further purified using chromatographic techniques. The synthesisof naturally occurring curcuminoids and related compounds is well knownin the art. The skilled artisan is referred to e.g., Pedersen, et al.,Ann. Chem., 1557-69, 1985; Arrieta et al., J Prakt. Chem., 334:656-700,1991 and Roughly et al, JCS Perkins Trans I, I, 2379-88, 1973, forguidance regarding detailed description of such synthesis andcharacterization.

Methods of Detection and Purification

The present invention concerns the provision, for example, as dietarysupplements of a number of fatty acyl compositions. The fatty acidmetabolism in circulatory and neutrophil cells has a balance ofdifferent precursors and substrates of arachidonic acid metabolism. Inproviding exogenous fatty acids as dietary supplementation, thisbaseline balance of fatty acids likely is altered. In certain instancesit may be necessary to monitor the levels of the different fatty acidspresent in an individual's circulation and/or neutrophils. The presentinvention encompasses methods for the determination of the fatty acylcontent of cells. These methods can also be employed for purifying fattyacids for inclusion as part of a dietary supplement. Generally, thesemethods will follow the methods described in the examples of the initialcharacterization of lipid content.

Chromatographic Methods of Detection

Briefly, one generally will isolate the lipid components of a cell asdescribed herein. Separation of lipid components from (i) non-lipidcomponents and (ii) each other will then permit quantitation of thedifferent lipid species. Quantitation of separated components may beachieved by any standard methodology, that would includephotodensitometric scanning of TLC plates or scintillation counting ofmembrane bound or liquid samples separated by various chromatographictechniques.

Any of a wide variety of chromatographic procedures may be employed. Forexample, thin layer chromatography, gas chromatography, high performanceliquid chromatography, paper chromatography, affinity chromatography orsupercritical flow chromatography may be employed. See Freifelder,Physical Biochemistry Applications to Biochemistry and MolecularBiology, 2^(nd) ed. Wm. Freeman and Co., New York, N.Y., 1982.

Partition chromatography is based on the theory that, if two phases arein contact with one another, and if one or both phases constitute asolute, the solute will distribute itself between the two phases.Usually, partition chromatography employs a column that is filled with asorbent and a solvent. The solution containing the solute is layered ontop of the column. The solvent is then passed through the columncontinuously, which permits movement of the solute through the columnmaterial. The solute can then be collected based on its movement rate.The two most common types of partition chromatography are paperchromatography and thin-layer chromatography (TLC); together these arecalled adsorption chromatography. In both cases, the matrix contains abound liquid. Other examples of partition chromatography are gas-liquidand gel chromatography.

Paper chromatography is a variant of partition chromatography that isperformed on cellulose columns in the form of a paper sheet. Thistechnique may be useful in identifying and characterizing the lipidcontent of a particular sample. Cellulose contains a large amount ofbound water even when extensively dried. Partitioning occurs between thebound water and the developing solvent. Frequently, the solvent used iswater. Usually, very small volumes of the solution mixture to beseparated are placed at the top of the paper and allowed to dry.Capillarity draws the solvent through the paper, dissolves the sample,and moves the components in the direction of flow. Paper chromatogramsmay be developed for either ascending or descending solvent flow. Twodimensional separations are permitted by changing the axis of migration90° after the first run.

Thin layer chromatography (TLC) is very commonly used to separate lipidsand, therefore, is considered a preferred embodiment of the presentinvention. TLC has the advantages of paper chromatography, but allowsthe use of any substance that can be finely divided and formed into auniform layer. In TLC, the stationary phase is a layer of sorbent spreaduniformly over the surface of a glass or plastic plate. The plates areusually made by forming a slurry of sorbent that is poured onto thesurface of the gel after creating a well by placing tape at a selectedheight along the perimeter of the plate. After the sorbent dries, thetape is removed and the plate is treated just as paper in paperchromatography. The sample is applied and the plate is contacted with asolvent. Once the solvent has almost reached the end of the plate, theplate is removed and dried. Spots can then be identified byfluorescence, immunologic identification, counting of radioactivity, orby spraying varying reagents onto the surface to produce a color change.

In Gas-Liquid chromatography (GLC), the mobile phase is a gas and thestationary phase is a liquid adsorbed either to the inner surface of atube or column or to a solid support. The liquid usually is applied as asolid dissolved in a volatile solvent such as ether. The sample, whichmay be any sample that can be volatized, is introduced as a liquid withan inert gas, such as helium, argon or nitrogen, and then heated. Thisgaseous mixture passes through the tubing. The vaporized compoundscontinually redistribute themselves between the gaseous mobile phase andthe liquid stationary phase, according to their partition coefficients.

The advantage of GLC is in the separation of small molecules.Sensitivity and speed are quite good, with speeds that approach 1000times that of standard liquid chromatography. By using a non-destructivedetector, GLC can be used preparatively to purify grams quantities ofmaterial. The principal use of GLC has been in the separation ofalcohols, esters, fatty acids and amines.

High Performance Liquid Chromatography (HPLC) is characterized by a veryrapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

Affinity Chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculethat it can specifically bind to. This is a receptor-ligand typeinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (alter pH, ionic strength, temperature, etc.). The matrixshould be a substance that itself does not adsorb molecules to anysignificant extent and that has a broad range of chemical, physical andthermal stability. The ligand should be coupled in such a way as to notaffect its binding properties. The ligand should also provide relativelytight binding, and it should be possible to elute the substance withoutdestroying the sample or the ligand. One of the most common forms ofaffinity chromatography is immunoaffinity chromatography. The generationof antibodies that would be suitable for use in accord with the presentinvention is discussed below.

Pharmaceutical Compositions and Routes of Administration

The nutritional compositions of the present invention will have aneffective amount of a Δ⁵ desaturase inhibitor, optionally an ω-3competitive inhibitor of AA metabolism such as stearidonic acid, andGLA, alone or in combination with other dietary supplements. Suchcompositions will generally be dissolved or dispersed in an acceptablecarrier or medium, preferably for oral or topical administration. Incertain embodiments, the compositions may be formulated for intravenous,intraarterial, intramuscular, nasal, vaginal, or anal administration,however, in certain embodiments the preferred medium is a milk-based orjuice based liquid.

The phrases “pharmaceutically or pharmacologically acceptable” refer tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, orhuman, as appropriate. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredients, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients, such as other fatty acidsupplements, vitamins, minerals, non-steroidal anti-inflammatories, etc.can also be incorporated into the compositions.

The compounds are generally formulated for oral administration. Suchpharmaceutically acceptable forms include, e.g., capsules, particularlygel capsules, or any other form currently used, including cremes, andliquids, for example syrups, suspensions or emulsions, inhalants and thelike.

A liquid formulation will generally consist of a dispersion of the fattyacid compositions in a suitable liquid carrier(s) for example, waterand/or other solvents such as, for example, polyethylene glycols, oils,milk, phospholipids, with, in certain formulations, a suspending agent,emulsifier, preservative, anti-oxidant, flavoring, and/or coloringagents. Preferred ingredients may include any of the following:galactolipids, sphingolipids, lecithins, cellulose, malt or maltextract, gelatin, casein, cholesterol, egg yolk, sodium dodecyl sulfate,benzalkonium chloride, p-hydroxybenzoic acid, vitamin C, vitamin E oralpha-tocopherol. A composition in the form of a dried powder may beprepared using any suitable pharmaceutical carrier(s) routinely used forpreparing solid formulations. Examples of such carriers includemagnesium stearate, starch, lactose, sucrose and cellulose.

A composition in the form of a capsule can be prepared using routineencapsulation procedures. For example, a dispersion or suspension can beprepared using any suitable pharmaceutical carrier(s), for exampleaqueous gums, celluloses, silicates or oils and the dispersion orsuspension then filled into a soft gelatin capsule.

Preferably the composition is in unit dose form such as a tablet,capsule, canned drink, or powder. Each dosage unit for oraladministration contains preferably from about 1 to about 15 g of GLA andbetween about 0.1 and 10 g of EPA or a pharmaceutically acceptable saltthereof calculated as the free base.

The pharmaceutically acceptable compounds of the invention will normallybe administered to a subject in a daily dosage regimen. For an adultpatient this may be, for example, an oral dose of GLA between 1 gram and15 grams, preferably between 1 gram and 10 grams, and most preferablybetween 1.5 grams and 3 grams, an oral dose of EPA between 0.1 g and 10grams, preferably between 0.25 grams and 5 grams and most preferablybetween 0.5 grams and 3 grams, and optionally an oral dose of SA betweenabout 0.1 g and about 15 g. The pharmaceutical compositions may beadministered 1 to 4 times per day. Thus in particular embodiments,compositions are contemplated comprising a 1:1 (w/w) ratio of GLA: EPA,wherein there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 grams of GLA. Inother embodiments there may be a 2:1 ratio of (w/w) ratio of GLA:EPA,wherein there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 14 or 15grams of GLA. Of course, the ratio of GLA:EPA administered may be variedfrom that disclosed herein above, however, it is desirable to includethe lowest effective amount of EPA or other fish-derived oils because ofundesirable odors and flavors associated with those oils. For example,any amount of EPA including 0.1, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or10 grams of EPA may be administered with any amount of GLA including 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 grams of GLA. Suchamounts of either supplement may be admixed in one composition or may bein distinct compositions.

The preparation of a composition that contains the Δ⁵ desaturaseinhibitor (EPA), stearidonic acid, and GLA compounds alone or incombination with other supplements as active ingredients will be knownto those of skill in the art in light of the present disclosure.Typically, such compositions can be prepared as liquids for capsules;solid forms or suspensions; the preparations can also be emulsified.

The dietary supplement comprising the combined Δ⁵ desaturase inhibitorand GLA formulations of the present invention may be in the form ofingestible liquids. For example, European patent application number EP0713 653 A1 and EP 0711 503 A2 (incorporated herein by reference)describe fruit juices and milk based liquids that can be fortified withGLA and other dietary supplements. In alternative embodiments, thecombined Δ⁵ desaturase inhibitor and GLA formulations of the presentinvention may be incorporated into a dietary margarine or otherfoodstuff.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in liquid suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereof,and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical formulations suitable for ingestion may includesesame oil, evening primrose oil, peanut oil, aqueous propylene glycol,and sterile powders. In all cases it is desirable to keep theformulation sterile and stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi.

The active compounds may be formulated into a composition in a neutralor salt form. Pharmaceutically acceptable salts, include the acidaddition salts and those which are formed with inorganic acids such as,for example, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts can also bederived from inorganic bases such as, for example, sodium, potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, histidine, Procaine and the like.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion, and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe compositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

Sterile compositions are prepared by incorporating the active compoundsin the required amount in the appropriate solvent with various of theother ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient.

Upon formulation, the active ingredients will be administered in amanner compatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms, such as tablets containing measured amounts ofactive ingredient, with even drug release capsules and the like beingemployable. The amounts of active ingredients in the formulations of thepresent invention will be similar to fatty acid supplements currentlyavailable. Those of skill in the art are referred to the Physicians DeskReference for more comprehensive details on currently used dosages offood supplements. Some variation in dosage will necessarily occurdepending on the condition of the subject being treated. The personresponsible for administration will, in any event, determine theappropriate dose for the individual subject.

For dietary or nutriceutical use, an inhibitor of Δ⁵ desaturase, aloneor in combination with other dietary supplements may be formulated intoa single or separate pharmaceutically acceptable compositions.Preferably formulations include a good tasting, milk based drink, or agood tasting, juice based drink or fruit based powder. Such a drink maybe contained in cans, preferably cans sealed under nitrogen or otheroxidatively inert gas atmosphere. Cans may be packaged in “six packs”held together by plastic or cardboard containers for easy retail sales.The drinks may also be enclosed in individual cardboard or aluminumbased or other foil containers, for example, that also provide a strawfor each individual container. The drink formulations may also beprovided in dried or lyophilized forms for rehydration in milk, water,juice, or other suitable solvent. In certain embodiments, a pre-measuredliquid container indicating the level of liquid needed for properrehydration may be included, and in bulk powder containers, a measuringspoon may also be provided. It is also understood that individualpackets may be provided that each include enough powder for a singleserving.

The following described materials and methods were used in the studiesdescribed in the Examples, below, unless otherwise indicated.

Cell and Serum Preparations

Neutrophils are obtained from venous blood of healthy human donors asdescribed (Lykens et al., Am. J. Physiol, Lung Cell Mol. Physiol.,262:L169-L175, 1992).

Eosinophils are purified by negative, immunomagnetic selection usingmonoclonals against FcKRIII (CD 16) present on neutrophils. Antibodytagged neutrophils are then incubated with anti-mouse IgG conjugatedmagnetic beads and removed by filtration over a magnetized steel woolcolumn.

Monocytes are obtained as follows: a mononuclear cell layer is obtainedfrom normal human blood after centrifugation over isolymph and washed inHBSS without Ca²⁺ or Mg²⁺, with 0.1% gelatin and 2 mM glucose, pH 7.4.Mononuclear cells are further separated by centrifugation overdiscontinuous Percoll gradients (45°/50.5%, 15 min, 300×g) to obtain arough separation of monocytes from lymphocytes, washing, and thencentrifugation over 48% Percoll (15 min, 300×g) to remove contaminatinglymphocytes.

In order to obtain Alveolar Macrophage (AM), BAL fluid samples arestrained through a monolayer of coarse mesh surgical gauze and totalcell counts and differentials determined. Cells are pelleted,resuspended in PBS, and washed 3 times. In normal individuals, ˜1 to1.5×10⁷ total cells are expected with ˜85% of harvested cells being AM.When necessary (to attain a population of at least 85% AM), AM arefurther purified by centrifugation (300×g, 15 min) over 48% Percoll.Cells are washed (3× in buffer and resuspended at 1×10⁷ cells/ml inHBSS.

Serum is extracted from 2 ml of venous blood from donors. Briefly, bloodsamples are incubated at 37° C. for 30 min. Blood clots are removed fromthe serum by centrifugation (600×g, 10 min). Residual red blood cellsare removed from the serum by centrifugation using a Beckman Microfuge Efor 5 min. After the addition of 1.9 ml of water to a 0.1 ml aliquot ofthe serum, lipids are extracted by the method of Bligh and Dyer (Blighand Dyer, Can. J. Biochem. Physiol., 37:911-920, 1959); Chilton et al.,J. Biol. Chem., 258:7268-7271, 1983). A portion (5%) of the extractedlipids is used to determine the mole quantities of fatty acids by GC/MS.Serum components are isolated into individual glycerolipid classes byTLC (System II or normal phase HPLC (Bligh and Dyer, 1959).

Chromatography Techniques

Phospholipid classes (PE, PS, PL and PC) are separated by normal phaseHPLC using an Ultrasphere-Si column (4.6×250 mm) eluted initially withhexane:2-propanol:ethanol:25 mM phosphate buffer (pH 7.4):acetic acid(490:367:100:30:0.6, v/v) at a flow rate of 1 ml/min. After 5 min, thecomposition of the phosphate buffer is increased to 5% over a 10 minperiod to elute all phospholipids.

TLC of phospholipid subclasses. Phospholipid subclasses (diacyl-,alkylacyl-, alky-1-enylacyl-) are separated as diglyceride acetates orbenzoates on silica gel G plates developed in benzene/hexane/ether(50:45:4, v/v). Briefly, the phosphobase moiety of phospholipids isremoved by phospholipase C hydrolysis followed by the addition of aceticanhydride/pyridine (5:1, v/v).

Leukotrienes are separated by reverse phase HPLC utilizing anUltrasphere ODS column (2.1 mm.×250 mm: Rainin Instrument Co, Woburn,Mass.) eluted with methanol/water/phosphoric acid (550:450:0.2 v/v, pH5.7) as the mobile phase at 0.3 ml/min. After 5 min the methanolcomposition of the mobile phase is increased from 55% to 100% over a 20min period. The mole quantities of each leukotriene are determined byexamining its UV optical density at 270 nm. Individual peaks areintegrated and their recoveries normalized by comparing these integratedareas to that of PGB₂ added as an internal standard.

GC/MS Analysis of Fatty Acids and Lipid Mediators

Free fatty acids are obtained from glycerolipids by base hydrolysisusing 2 N KOH (30 min, 60° C.). After the addition of an equal volume ofwater, the pH of the reaction mixture is adjusted to 3 using 6 N HCl.Free fatty acids are then extracted with ethyl ether and converted topentafluorobenzylesters using an equal volume of 20%pentafluorobenzylchloride in acetonitrile and 20% diisopropylethylaminein acetonitrile. The carboxylate anion of all fatty acids of interestand [²H₃]-stearic acid and [²H₈]-arachidonate (internal standard) areanalyzed by NICI GC/MS using a Hewlett Packard mass spectrometer (HP5989A).

Eicosanoids from ethyl acetate extracts of supernatant fluids areconverted to methoxime-pentafluorobenzyl ester trimethylsilylderivatives. These derivatives of LTB₄, LTB₅, ²H₄-LTB₄, PGE₂, PGE₁, ²H₄PGE₂ and H₄ PGE₁ (internal standard) are analyzed on an HP selectivemass detection system (Hewlett Packard 5989A) by selected ion monitoringtechniques to record carboxylate anions at m/z 479, 477, 483, 524, 526,528 and 530, respectively.

Urinary LTE₄

Aliquots of urine are spiked with ³H-LTE₄ and stored at −70° C. UrinaryLTE₄ is then measured using the methods of Manning et al., J. AllergyClin. Immun., 86:211-220, 1990 utilizing reverse phase HPLC followed byRIA: (Christie, J. Lipid Res., 26:607-612, 1985). Recovery is determinedusing the added [³H]-LTE₄ as an internal standard. LTE₄ levels areexpressed relative to urinary creatinine.

Subjects and Controlled Diets

Subjects are recruited by poster advertisements from the Medical Centerstaff and students. Inclusion criteria require healthy, normal men andwomen of all races, 21 to 55 years old; subjects who consume anomnivorous, nutritionally adequate diet consisting of at least 25% ofcalories from fat. Volunteers who are within 10% of ideal body weight(IBW) and do not exceed 30% and 35% body fat for men and women,respectively (as determined by anthrometric measurements in the GCRC).Diet compositions are determined by the food frequency questionnairecomponent of the Health Habits and History Questionnaire developed bythe NCl (Shin et al., Am. J. Respir. Crit. Care Med., 149:660-666, 1994;Wenzel et al., Am J. Respir. Crit. Care Med., 156:737-743, 1997).

Exclusion criteria include persons with any chronic or acute disease asdetermined by self report or physical screening; who are vegetarians orvegans; who are lactose or egg intolerant; who use drugs that affect AArelease and subsequent metabolism (steroidal and non-steroidalanti-inflammatories); with serum cholesterol levels above 220 mg/cd; whoare unable or unwilling to strictly adhere to a precise, restricteddiet; who are unwilling to be randomly assigned to the diet group forwhatever protocol the subject volunteers; who are smokers.

Composition of the diets are based on the USDA Handbook 8 and TheNutrition Data System from The Nutritional Coordinating Center of theUniversity of Minnesota. For each of the protocols outlined above, themenus are designed with adjustments for each subject's energy needs.Basal energy expenditure is determined by the Harris-Benedict Equations:Basal energy expenditure (BEE) for men=65+(13.7×Wt(kg))+(5×Ht(cm))−(6.8×age(yr))for women=655+(9.6×Wt(kg))+(1.8×Ht(cm))−(4.7×age(yr)).

Total daily energy needs equal the BEE times an activity factor of from1.3 for ambulatory but sedentary to 1.5 for the more active persons.Body weight is monitored each day when the subjects come to the Centerto receive their meals. Calorie levels are adjusted appropriately.

Procedures and Specimens Collection used in Human Model of Atopic Asthma

Clinical data on each patient is entered into a database consisting ofthe following elements. Demographic data (age, sex, race, smokinghistory), and the data elements used to fulfill the above diagnosticcriteria, spirometric data, presence of atopy (positive “prick” skintesting to respirable antigens), presence of LAR to inhaled antigen, andpresence of allergic rhinoconjunctivitis.

-   Allergen skin testing—Atopic asthmatic subjects are identified by    skin testing using the skin prick method at a 1:10 (wt/vol) dilution    of 20 stock antigen solutions (Greer Laboratories, Lenoir, N.C.).    Subjects must not be receiving immunotherapy, nor may they be    treated with systemic corticosteroids for a minimum of 4 wk. Short    acting antihistamines are avoided for at least 24 h and long acting    for at least 7 days. Atopic subjects are defined as those with a    positive response consisting of a wheal of at least 3 by 3 mm to one    or more antigens, with an appropriately negative saline control.-   Allergen Inhalation Challenge—The immediate (early) asthmatic    response (EAR) or late asthmatic response (LAR) is studied under    controlled conditions using inhaled antigenic challenge in volunteer    patients with asthma using a previously described protocol (Smith et    al., Clin. Pharm. Ther., 54:430-436, 1993). Atopic asthmatics    undergo inhaled allergen challenge followed by BAL according to the    following protocol. Subjects must have no lung disease other than    asthma, and, on the day of testing, must have a baseline FEV₁>70% of    predicted. Subjects must not be receiving immunotherapy, nor may    they be treated with cromolyn sodium or corticosteroids (inhaled or    systemic) or leukotriene antagonist for a minimum of 4 wk.    Short-acting antihistamines are avoided for at least 24 h and    intermediate acting for 7 days (astemizole for 6 weeks).    Theophylline preparations are withheld for 24 h prior to challenge    and beta-agonists for 8 h prior to challenge. On the day of    challenge, subjects must be wheeze-free, with an FEV₁>than 80% of    the previously observed highest value. If the patient has an    intercurrent respiratory infection, inhalation challenge is    postponed for at least 6 wk. Antigens to which the subject is    perennially exposed (e.g., mite, cat) are utilized whenever possible    to minimize the impact of seasonal variations in environmental    exposure to the specific antigen. Further, antigen testing is    conducted out of the respective allergen season, or after attempts    to minimize environmental exposure (e.g., to mites or cats) have    been implemented. Antigenic challenge generally begins between 7:30    and 8:00 am and the patient is monitored for a minimum of 12 h    following antigenic challenge. Subjects inhale allergen to which    they have previously demonstrated skin sensitivity beginning at    1:1,000,000 dilution (wt/vol) and proceeding with logarithmically    increasing concentrations to 1:100. The subject breathes quietly    from a continuous hand-held nebulizer for 2 min at each    concentration. Following each concentration, the FEV₁ is measured at    5 min intervals (DS Plus, Warren E. Collins, Inc., Braintree,    Mass.). If the FEV₁ does not fall by 20% after 15 min, the next    higher concentration is administered. Once a 20% drop in FEV₁ is    measured, or following the highest concentration, spirometry is    performed every 15 min for the first hour and then hourly for the    next 11 h. Patients experiencing symptomatic bronchospasm following    initial antigenic inhalation may receive a short acting inhaled    beta-agonist bronchodilator agent (isoproterenol). Thi S, has no    effect on the subsequent late asthmatic response (LAR). An LAR is    defined as a 15% or greater fall in FEV₁ from the prechallenge    baseline value occurring between 3 to 12 h after challenge.-   Bronchoalveolar lavage—Subjects whose FEV₁ immediately preceding    bronchoscopy is less than 60% of prechallenge baseline do not    undergo BAL to minimize further acute diminution of lung function    and to maximize subject safety. Fiberoptic bronchoscopy is performed    following methodologies previously detailed in the literature    (Wenzel et al., J. Allergy Clin. Immunol., 87:540-548, 1991; Zehr et    al., Chest, 95:1059-1063, 1989). Briefly, the fiberoptic    bronchoscope is introduced into the lower airways trans-nasally    following nebulized 4% Xylocaine, topical anesthesia and    benzodiazepine sedation, titrated to patient comfort Isoproterenol,    1 puff, 130 μg is administered 10 min before bronchoscopy.    Bronchoalveolar lavage (BAL) is obtained from the right middle lobe    or lingula utilizing six 50 ml aliquots (200 ml total volume) of    sterile normal saline—without preservatives, warmed to 37° C. The    amount of BAL returned is recorded and the specimen promptly    processed. The right middle lobe or lingula is routinely used to    maximize the uniformity of specimen yield as the return from BAL is    dependent upon many factors, but especially airway geometry and    gravity. These areas tend to drain spontaneously by gravity in    supine patients. This improves the return of fluid from the lavage    as well as minimizing the amount of retained fluid within the lung    in these patients.

BAL samples are strained through a monolayer of coarse-mesh surgicalgauze and total cell yield determined by taking a small aliquot of thepooled, well mixed fluid, and counting the cells in a Neubauerhemocytometer. BAL cell count is expressed as the total number of cellsrecovered by lavage and as the number of cells per ml of recovered BALfluid. A small aliquot is then cytocentrifuged (Shandon SouthernCytospin) for 5 min at 4,500 RPM, air dried, and stained by a modifiedWright-Giemsa stain. A 300 cell differential count is performed wherealveolar macrophages and other leukocytes are enumerated. The number ofciliated or squamous epithelial cells present are noted, but are notincluded in the differential count. Quantification of the cellularpopulations recovered by lavage are expressed as a percentage of thetotal cells recovered (excluding red blood cells and epithelial cells),and as the total numbers of each cell type recovered. The remaining BALfluid is centrifuged at 500×g, 4° C., for 15 min. Aliquots of thesupernate not immediately processed are stored at −80° C.

Effect of supplementation on eosinophil eicosanoid biosynthesis: 75 mlfrom a peripheral vein is collected 30 to 60 min prior to inhaledchallenge and 24 h after challenge. Eosinophils are isolated asdescribed above. Cells are challenged with A23187 (1 μM) and PAF (1 μM).Leukotrienes are quantified after reverse phase HPLC as described above.Quantities of free fatty acid and prostaglandins are determined by NICIGC/MS.

-   Urinary LTE₄: Urine is collected for 3 h beginning immediately after    antigen challenge and again from 3 h until after the LAR. Urinary    LTE₄ is measured using an RIA as described above.-   Arachidonic acid release: Free fatty acid levels including AA in BAL    are determined, after addition of ²H₃-stearidonic acid and ²H₈-AA to    BAL as internal standards, by NICI-GC/MS.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE 1 In Vivo Studies Examining GLA Supplementation in Humans

Incorporation of Supplemented Fatty Acids into Serum Lipids

Initial studies examined the effect of dietary supplementation with GLAon the fatty acid content of serum lipids. Here, 9 healthy adultvolunteers consumed a controlled eucaloric diet consisting of 25% fat,55% carbohydrate and 20% protein prepared in the metabolic kitchen ofthe GCRC.

Four menus were served on a rotating basis throughout the study period.In addition, three groups of three volunteers supplemented this dietwith three different doses of GLA.

FIG. 2 demonstrates the effect of GLA supplementation at three differentdoses on serum levels of GLA, DGLA, and AA. In all three groups ofsubjects, AA significantly increased in serum lipids at the end of thethree-week dietary period when compared with baseline values. Both GLAand DGLA significantly increased in the groups receiving 3.0 g/day and6.0 g/day. In the two highest dose groups, DGLA levels increasedtwo-fold and AA levels increased approximately 30% when compared tobaseline values of these fatty acids in the same subjects. There was nosignificant change in serum fatty acid levels of volunteers eatingcontrol (25% energy as fat) diets, but not receiving the GLA supplement.

An important difference between the aforementioned studies and mostclinical trials in the literature was the length of time ofsupplementation. Therefore, a long-term supplementation study (3.0g/day) was performed over a 12-week period to assess whether fatty acidratios and distribution would change in a manner that was not observedat three weeks. This study showed that there was a significant increasein serum GLA, DGLA, and AA levels by two weeks and that these levelsstayed high over an additional 10 weeks of supplementation (FIG. 3).Taken together, these data suggest that although some dietary GLAremains in the serum unchanged, substantial quantities of the elongationproduct (DGLA) and the elongation/Δ⁵ desaturase product (AA), accumulatein serum after GLA supplementation.

The next set of studies was designed to determine the distribution ofsupplemented fatty acids or their metabolites within individualglycerolipid classes of serum. Serum was collected from volunteersbefore and after receiving 6.0 g/day of GLA. Serum glycerolipids wereseparated by TLC and fractions were analyzed for fatty acid contentfollowing base hydrolysis by NICI-GC/MS. GLA was located predominatelyin triglycerides (36-38% of total), phospholipids (26-33% of total), andcholesterol esters (17-21% of total). After supplementation, GLAsignificantly increased in both phospholipids and cholesterol esters. Incontrast, DGLA and AA were almost exclusively located in serumphospholipids, with very little of these fatty acids found in otherserum pools. After supplementation, both DGLA and AA increased only inphospholipid pools.

Incorporation of Supplemented Fatty Acids into Neutrophil Lipids

The fatty acid composition of the neutrophil lipids in subjects eating acontrolled diet supplemented with 1.5, 3.0, or 6.0 g/day of GLA werealso analyzed. No consistently detectable amounts of GLA were found inthe glycerolipids of neutrophils before or after supplementation.Although relatively large quantities of AA were found in unsupplementedneutrophils, there was no significant change in AA within glycerolipidsafter supplementation at any of the doses given (FIG. 4). In contrast,DGLA within glycerolipids increased as a function of the dose providedto the volunteers. The AA/DGLA ratio decreased from approximately 5.4:1before supplementation to 2.3:1 three weeks after 6.0 g/day of GLAsupplementation. There was no significant change in fatty acid levels incontrol subjects eating the study diet without supplementation. Thesefindings suggest that neutrophils rapidly elongate GLA to DGLA but lackthe ability to desaturate DGLA to AA.

The influence of long term (12 week) GLA supplementation (3 g/day) onthe composition of GLA, DGLA and AA in neutrophil lipids also wasexamined. In contrast to serum, GLA supplementation resulted in anincrease in DGLA but not AA even at 12 weeks (FIG. 5). It is not clearwhy the increase of AA in serum is not eventually observed in neutrophillipids; perhaps this AA is in a serum pool not available to neutrophils.Taken together these preliminary data indicate that GLA provided as adietary supplement is converted to different products (DGLA ininflammatory cells and AA in serum) depending on where it ismetabolized. This results in the potentially beneficial effect ofreducing AA metabolism in inflammatory cells balanced against thepotential adverse effects of the accumulation of serum AA levels. Thesestudies led to studies designed to determine whether it is possible toutilize the endogenous elongase activity within inflammatory cells tosynthesize analogs of AA from appropriate dietary precursors withoutconcomitantly increasing levels of circulating AA.

To better determine the distribution of fatty acids within differentglycerolipid classes, neutrophils were obtained before and aftersupplementation with 6.0 g/day of GLA for 3 weeks and glycerolipids wereseparated by normal phase HPLC. Quantities of fatty acids in eachglycerolipid class were then determined by NICI-GC/MS. As shown in FIG.6A, the majority of AA (>60%) within the neutrophil lipids was locatedin phosphatidylethanolamine (PE) and neither the absolute amount nor itsrelative distribution changed significantly after dietarysupplementation with GLA. Similarly, the bulk of DGLA in the neutrophilwas associated with PE (40%) (FIG. 6B). There were significant increasesin the amount of DGLA associated with both PE and neutral lipids aftersupplementation, For example, the AA/DGLA ratio in PE decreased from8.3:1 before supplementation to 4:1 after supplementation. These dataillustrate that AA and DGLA reside in similar glycerolipid pools bothbefore and after supplementation.

Influence of GLA Supplementation on the Release of Fatty Acids or theProduction of Lipid Mediators by Stimulated Neutrophils

Neutrophils were next obtained from subjects before and aftersupplementation and stimulated with ionophore A23187. The release of AAfrom the neutrophil glycerolipids after stimulation did not changefollowing supplementation. However, the release of DGLA increased by63%, 65%, and 69% in those volunteers receiving 1.5 g, 3.0 g and 6.0g/day GLA, respectively (FIG. 7). These data support the hypothesis thatthe fatty acid composition of the neutrophil glycerolipids impacts onthe fatty acids released upon cellular stimulation. They also suggestthat the PLA₂ isotype(s) enzyme responsible for mobilizing fatty acidshydrolyzes DGLA in addition to AA.

While the aforementioned studies demonstrated that GLA supplementationdid not influence the ex vivo release of AA from neutrophilglycerolipids, it was unclear whether GLA supplementation would alterleukotriene biosynthesis. To examine this question, neutrophils werestimulated and the synthesis of LTB₄, 20-OH LTB₄, and the 6 transisomers of LTB₄ were measured by reverse phase HPLC analysis.Neutrophils from subjects supplementing their controlled diets with 3.0g/day GLA produced 60% less LTB₄ than the same subjects beforesupplementation (FIG. 8). 20-OH LTB₄, 6-trans LTB₄ and 6-trans 12-epiLTB₄ levels were decreased to a similar degree after supplementation.

A final set of studies measured changes in the capacity of neutrophilsto generate PAF ex vivo before and after GLA supplementation.Neutrophils of subjects receiving 3.0 g/day of GLA produced 40% less PAFafter supplementation than neutrophil obtained from those same subjectsbefore supplementation. Taken together, these data reveal that GLAsupplementation can alter the capacity of neutrophils to generate lipidmediators. This inhibition appears to occur at some step distal to thephospholipase-catalyzed cleavage of AA from membrane phospholipids.

EXAMPLE 2 In Vitro Studies Examining the Metabolism of GLA in HumanNeutrophils

It is generally assumed that the liver has a key role in the in vivoelongation and desaturation of n-6 fatty acids. However, the role ofother cells (especially inflammatory cells) and tissues has not beenextensively studied. In addition, it is critical to evaluate themechanism of leukotriene inhibition in less complex (than in vivo model)systems. To begin to address these problems, the inventor developed amodel in which neutrophils could be incubated long-term with fatty acidsor other fatty acid derivatives. Human neutrophils have been isolatedand cultured overnight in RPMI, 2% insulin-transferrin and fetal bovineserum (FBS). In initial studies, varying concentrations of GLA(complexed to albumin) were provided to these cultured neutrophils for24 h. FIG. 10 shows quantities of DGLA and AA in neutrophils atincreasing concentrations of GLA. The quantity of DGLA in neutrophilglycerolipids increased as a function of the concentration of GLA. Incontrast, there was no change in the quantity of AA in neutrophilphospholipids. These data revealed that neutrophils have the capacity totake up GLA and rapidly elongate it to DGLA. However, they do notdesaturate DGLA to form AA. These data are consistent with in vivofindings that indicate that GLA supplementation leads to an increase inDGLA, but not GLA or AA in human neutrophil glycerolipid. Furthermore,they provide direct evidence that the neutrophils themselves canelongate GLA in vivo.

It has been long recognized that arachidonate is hydrolyzed frommembrane glycerolipids by phospholipase A₂ isotypes during cellstimulation. However, to date, there is little direct evidence thatsimilar mechanisms exist to mobilize DGLA. To examine this question,neutrophils that had been cultured with varying concentrations of GLA (0to 200 nmol) were stimulated with ionophore A23187, and mobilized fattyacids were measured by NICI GC/MS. DGLA along with AA were released fromneutrophils during stimulation. To determine if neutrophils can furthermetabolize DGLA to oxygenated products, stimulated cells were provided[¹⁴C]-DGLA and products were measured by reverse HPLC. Neutrophilsprimed with LPS followed by stimulation with FMLP also converted DGLA to15 HETrE. FIG. 11 illustrates that A23187 stimulated neutrophils producea labeled product that migrated with 15-HETrE. In contrast, none of thisproduct was observed in unstimulated cells. To the inventor's knowledge,these are the first studies to demonstrate the capacity of neutrophilsto release DGLA and convert it into oxygenated products.

It is contemplated that neutrophils may also produce 8-hydroxy-9,11,14eicosapentaenoic acid from DGLA. Borgeat and colleagues reported this tobe a product of the incubation of dihomogammalinolenic acid with rabbitneutrophils. Studies were also designed to examine whether 15-HETrEproduced by neutrophils might influence LTB₄ generation. Previousstudies by Vanderhoek and colleagues have demonstrated that the AAproduct, 15-HETE, can reduce 5-lipoxygenase activity (Vanderhoek et al.,J. Biol. Chem., 255:10064-10066, 1980). Neutrophils were isolated fromnormal unsupplemented volunteers and were treated with variousconcentrations of 15-HETrE and then stimulated with ionophore A23187.FIG. 12 shows the generation of LTB₄ and its major metabolite 20-OH LTB₄by stimulated neutrophils. 15-HETrE induced a dose dependent inhibitionof leukotriene generation with an IC₅₀ of approximately 5 μM. Inaddition, DGLA at higher concentrations (IC₅₀, ˜10 μM) also inhibitedleukotriene generation. Although these studies do not prove that15-HETrE or DGLA is the in vivo inhibitor of 5-lipoxygenase, they revealthat DGLA and oxygenated products of DGLA can potently influenceeicosanoid generation.

EXAMPLE 3 Influence of the Combination of GLA And Eicosapentaenoic Acid(EPA) on the Fatty Acid Composition of Serum and Neutrophil Lipids

As mentioned above, a concern with the long-term effects of GLAsupplementation is that there is an increase in serum levels of AA.There is a need, therefore, to find dietary strategies that will producenatural antagonist of AA in inflammatory cells without increasing serumAA. Previous in vitro studies in isolated hepatocytes and in vivostudies in animals suggest that EPA is a product inhibitor of the Δ⁵desaturase (Gronn et al., Biochim. Biophys. Acta, 1125:3543, 1992; Danget al., Lipids, 24:882-889, 1989). In order to determine whether EPAwould perform a similar function in humans in vivo, three subjects oncontrol diets (25% fat) were supplemented with a combination of EPA (1.5g/day) and GLA (3.0 g/day) for three weeks. It was shown (FIG. 4 andFIG. 5) that this quantity of GLA (alone) induces marked increases inserum AA in both the short (3 weeks) and long term (12 weeks). Thecombination of GLA and EPA resulted in marked increases in GLA, DGLA andEPA in serum lipids. However, in contrast to the GLA supplementationalone, the combination of EPA with GLA did not cause an increase inserum AA (FIG. 13). These interesting results suggest that it may bepossible to block the Δ⁵ desaturase in humans with EPA thereby providinga means to supplement humans with high levels of GLA without concomitantincreases in serum AA levels.

EXAMPLE 4 In vitro Studies Examining the Metabolism of Stearidonic Acidin Human Neutrophils

As described above, human neutrophils (in vitro in overnight culture)will take up GLA and elongate it to DGLA but not further desaturate thatDGLA to AA. An alternative route to depleting AA in neutrophils may alsobe useful in modulating the inflammatory responses mediated by AA andits metabolites. It was contemplated that the n-3 fatty acid,stearidonic acid (18:4) would also be elongated in neutrophils to formω-3 arachidonic acid (FIG. 1). Varying concentrations of stearidonicacid were provided to cultured neutrophils for 24 h. Lipids wereextracted and the quantities of fatty acids determined after basehydrolysis using GC/MS. There was no detectable (ω-3 arachidonic acid inneutrophils before supplementation (FIG. 14). However, addition ofstearidonic acid caused a dose-dependent increase in ω-3 arachidonicacid in glycerolipids of these cells. In contrast to this increase,there was no increase in the Δ⁵ desaturase product of ω-3 arachidonicacid, eicosapentacnoic acid, nor was there an increase in AA. Analogousto supplementation with GLA, these data reveal that neutrophils have thecapacity to take up stearidonic acid and rapidly elongate it to ω-3arachidonic acid. However, they do not further desaturate ω-3arachidonic acid to form eicosapentaenoic acid.

These studies raise the interesting possibility that high levels of theAA analog, ω-3 AA, can be induced in inflammatory cells by providinginflammatory cells (in vitro or in vivo) with stearidonic acid.Moreover, they point out the potential for (ω-3 AA to compete withnatural AA (n-6) for enzymes (phospholipase A2 isotypes, cyclooxygenaseisotypes, and 5-lipoxygenase) that convert AA to oxygenated metabolites.

EXAMPLE 5 Development of A Model to Study the Influence of Diet onClinical And Biochemical Parameters of Asthma

Asthma presents a defined inflammatory disease that can be used as amodel to test the efficacy of dietary manipulation. To this end anasthma model in humans was developed to test the reproducibility of thein vitro data and to determine the best dietary strategies. Anotherbenefit of such a model is it allows the investigator to establish theeffect of antigen challenge on AA levels in bronchoalveolar lavage fluid(BALF). Thus the present example teaches the use of an asthmatic modelto test these parameters.

To this end, measures of airway physiology and analysis of BALF cellularand biochemical constituents were obtained from 5 stable atopicasthmatics before and after antigen challenge, both with and withoutprior corticosteroid therapy. A systemic corticosteroid arm was felt tobe an impost to validate the physiologic variables as well as toascertain which components in the BALF were sensitive markers ofsteroid-responsive inflammation. Additionally, AA levels were measuredin BALF 4 h after inhaled antigen challenge (7 subjects) and at the timeof the LAR (5 subjects). For comparison, identical BALF analyses wereperformed in ten normal volunteers (without antigen challenge orcorticosteroids).

Study Design

Asthmatic subjects were defined using criteria proposed by the AmericanThoracic Society, Am. Rev. Respir. Dis., 136:225-244, 1987. Normalsubjects were healthy non-smokers, without respiratory symptoms. In allsubjects, demographic data, history and physical examination, baselinespirometry, skin testing and methacholine PC20, using a tidal breathingtechnique, were obtained after informed consent for study participation.This was followed, no earlier than 7 days later, by baselinebronchoscopy for collection of BALF. This concluded the study protocolfor normal subjects.

In 5 subjects, inhaled antigen challenge was performed using apreviously described protocol and physiologic data collected. Not lessthan 2 weeks later, PC20 was again determined and antigen challengerepeated with BALF collected at the time of the LAR as determined duringthe first challenge. Two to 4 weeks later, these subjects were placed on40 mg of prednisone daily for 7 days. Inhaled antigen challenge wasagain performed and BALF obtained at the same time after antigenchallenge as on the previous visit. In an additional 7 subjects, BALFwas obtained 4 h after inhaled antigen challenge, but without asubsequent course of prednisone therapy.

Statistical Analysis

In the asthmatic patients, the changes in cell composition, eosinophilcationic protein (ECP), and protein in BALF among study conditions wereexamined using one way ANOVA with study period as the independentvariable. If a significant interaction was found, a paired t-test wasused to compare the means among test periods. Because the AA levels werenot normally distributed within the groups, the non-parametric Wilcoxonsigned-ranks test was used to analyze the differences in thesemeasurements. A p<0.05 was used to determine statistical significance.

Results

Measures of airway response to antigen challenge were consistent andreproducible both immediately and at LAR. The mean time for LAR, was6.4±1.5 h after challenge. The mean (±SD) fall in FEV₁ immediately afterantigen challenge was 35±8% while the fall at LAR was 28±18% frombaseline FEV₁. Following prednisone, both the immediate response and LARwere ablated.

The percentages of neutrophils and eosinophils in BALF weresignificantly higher in the asthmatics. The level of ECP rose afterantigen challenge and was suppressed by corticosteroid administration(p=0.03). While the percentage of eosinophils tended to mirror thechanges in ECP, these changes did not achieve statistical significance.AA levels in BALF rose after antigen challenge (mean±SE:baseline=2.2±0.3 ng/ml BALF; post-challenge=3.9±1.0; p<0.05).

Discussion

This antigen challenge model of asthma provides reproducible physiologic(pulmonary function) data within and between subjects. Further, ECPappears to be a reproducible surrogate measure of eosinophil presenceand/or activity in this model. In addition, AA levels can be observed toincrease after antigen challenge in this model. Collectively, thesemeasures offer the capability of assessing the efficacy of dietarymanipulation with the expectation that significant differences amongtreatment regimens can be detected with a relatively small number ofstudy subjects.

EXAMPLE 6 Effect of GLA Supplementation on Eosinophil Fatty AcidComposition and Airway Functions

An issue with the GLA data obtained with neutrophils is its relationshipto atopic asthma and, in particular, whether the neutrophil has a keyrole in atopic asthma. While there is evidence that the neutrophil has arole in atopic asthma, previous studies, to date, point to theeosinophil as having a central role. Therefore, it was important todetermine how GLA was metabolized by human eosinophils. Thus,eosinophils were isolated from atopic subjects and incubated with GLA asdescribed above. Like the human neutrophils, supplementation of humaneosinophils resulted in a marked increase in DGLA but no change in thequantity of AA in eosinophil glycerolipids (FIG. 15). These data revealthat eosinophils have the capacity to take up GLA and rapidly elongateit to DGLA. However, eosinophils do not further desaturate DGLA to formAA.

In a second set of studies, two atopic asthmatics were recruited andchallenged with antigens as described above. In both subjects, theconcentration of antigen necessary to drop FEV₁ by greater than 20% wasestablished. At a subsequent date, they were each challenged with theserespective concentrations of antigen and monitored with spirometry toassess the development of an early and late responses. Each of thesesubjects were then placed on GLA supplementation for four weeks and thenchallenged again with the same dose of antigen. The subjects were thenplaced back on their normal diets for two weeks and then challengedagain with the same respective dose of antigen. FIG. 16 shows theaverage of the responses of the two subjects at the three challengeperiods. The magnitude of the early response was diminished (whencompared to pre -and post GLA supplementation) in both subjects fourweeks after GLA supplementation. In contrast, GLA supplementation didnot influence the late response.

Additionally, the influence of GLA supplementation in a human model ofatopic asthma, on eicosanoid production, bronchial reactivity, andairway cellular influx can be measured as detailed herein. A randomorder, placebo-controlled crossover design preceded by a control diet“run in” phase study is performed. A crossover design is chosen to keepthe number of subjects required for statistical validity as small aspossible by minimizing the influence of intersubject variability withregards to the severity of asthma, environmental triggers and exposures,and nature and severity of the late asthmatic response (LAR). Subjectsare studied after 3 weeks of a controlled “normal” diet with 25% ofcalories from fat, after 3 weeks of the “experimental” diet consistingof the “normal” diet supplemented with 4.5 grams (15 capsules/day) ofGLA as borage oil, and after 3 weeks of a “placebo” diet consisting ofthe “normal” diet with 4.5 grams (15 capsules/day) of olive oil. Oliveoil is 70% oleic acid, 13% C16, and 15% C18, (<1%, n-3) fatty acids astriglycerides. Neither oil supplement has either an odor or a taste whenin capsule form. The experimental and placebo diets are given in randomorder. Each 3 week period is separated by a 4-6 week usual diet“washout” period when the diet of the study subjects is not controlled.Preliminary data from this group suggests that 4 weeks is a sufficienttime period for abolition of an effect of diet during fine precedingstudy period.

Results

It is contemplated that GLA supplementation and not placebo or “normal”diets will mitigate the response to antigen challenge as measured by thedecrements in FEV₁ both immediate and the LAP, and reduce the influx ofeosinophils into airways during the LAR. GLA supplementation will alsolikely attenuate antigen-induced urinary LTE₄ exertion and BALF AAincreases.

While the antigen challenge model is capable of detecting a therapeuticeffect due to prednisone with a small number of subjects, GLAsupplementation may be associated with smaller, though significant,effects that are overlooked using relatively small sample sizes. Thetrial uses 10 subjects per group. Sample sizes are based on varianceestimated and differences reported in the preliminary results. Thecontemplated sample sizes have a 90% power to demonstrate an effect onpulmonary function (FEVI) that is at least half the magnitude observedwith oral prednisone therapy in the pilot study, at an alpha of 0.05.Asthma is a complex disease process and it is possible that significanteffects in some components may be missed by using a model that is notsensitive to these effects. For example, an antigen challenge modelwould not be the appropriate system in which to detect an impact onneurally-mediated immediate processes (e.g., airway cooling). The effectof GLA supplementation, would, however, suggest that this antigenchallenge model is appropriate.

EXAMPLE 7 Dietary Strategies in Humans Utilizing Endogenous ElongaseActivity within Inflammatory Cells to Synthesize Structural Analogs ofAA from Dietary Precursors without Concomitantly Increasing Levels ofCirculating AA

The data suggest there may be two strategies that can be utilized inhumans to synthesize analogs of AA in inflammatory cells withoutconcomitant increases in serum AA. The first approach (FIG. 17A) is tosupplement the diets of humans with a combination of gammalinolenic acid(GLA) and a Δ⁵ desaturase inhibitor such as eicosapentaenoic acid (EPA),for example. This strategy is based on in vitro data in hepatocytes andin vivo data in animals which indicate that EPA is a product inhibitorof the enzyme activity that synthesizes it, the Δ⁵ desaturase (Gronn etal., 1992; Dang et al., 1989). The inventor has shown in two volunteersthat administering of GLA in combination with EPA will induce a markedaccumulation of DGLA in circulation and neutrophil lipids withoutcausing a marked accumulation of AA in serum lipids (which is seen withGLA supplementation in the absence of EPA).

If in vivo administration of EPA is an effective means to block thehepatic Δ⁵ desaturase, this combination should furnish a means toprovide high concentrations of GLA to humans to synthesize the closestructural analog of AA, DGLA, in inflammatory cells. This will have theaction of inhibiting AA metabolism and eicosanoid biosynthesis, andattenuating signs and symptoms of inflammatory disorders, without thesignificant side effect of the accumulation of AA in circulation.

The second approach involves administering the n-3 fatty acid,stearidonic acid, to humans (FIG. 17B). This fatty acid is converted (bythe endogenous elongase in inflammatory cells) to a structural analog ofAA, (ω-3 AA and this product will block AA metabolism and thus haveanti-inflammatory effects. There have been several studies over the lastfew years that have examined the effects of in vivo supplementation withalpha linolenic acid (18:3, n-3) in both humans and animals. Generally,these studies have shown that alpha linolenic acid has only modestanti-inflammatory effects (Nordstrom et al., Rheumatol. Int.,14:231-234, 1995; Larsson-Backstrom et al., Shock, 4:11-20, 1995; Clarket al., Kidney Int., 48:475-480, 1995; Shoda et al., J. Gastroenterol.,30(suppl 8):98-101, 1995). However, only a very small portion of alphalinolenic acid is converted to stearidonic acid by the Δ⁶ desaturase. Infact, this step appears to be the rate-limiting step in n-3polyunsaturated fatty acid biosynthesis. As described herein,stearidonic acid supplementation is an efficacious means to block AAmetabolism because it bypasses the rate-limiting step (Δ⁶ desaturase)and is directly utilized by inflammatory cell elongase activity. A majoradvantage of stearidonic acid verse GLA (alone) as a supplement is thatthe elongation/Δ⁵ desaturase product from this precursor is EPA and notAA. Consequently even if EPA accumulates in serum components, it willnot have the potential detrimental effects of AA.

EXAMPLE 8 Inhibition of Delta-5 Desaturase by Eicosapentaenoic Acid inHuman Liver Cells

The use of Δ⁵ desaturase inhibitors in the practice of the presentinvention rests, in certain aspects, on the ability of those inhibitorsto affect Δ⁵ desaturase activity in the hepatic cells of a subject whois receiving GLA or DGLA as a dietary supplement, or especially as atreatment for an inflammatory disorder or condition, for example. As isdescribed elsewhere herein, the DGLA, if taken up by liver cells, or GLAthat has been elongated to DGLA undergoes Δ⁵ desaturation in hepaticcells to produce arachidonic acid. This desaturation does not occur inimmune system cells such as neutrophils, which lack the Δ⁵ desaturaseactivity.

In order to demonstrate that an inhibitor such as eicosapentaenoic acidhas the capacity to block Δ⁵ desattirase, various concentrations of EPAwere added exogenously, along with DGLA, to a human liver cell line,HEP-G2, and the conversion of DGLA to AA was monitored. The results areshown in FIGS. 18A and 18B. The data indicate that EPA caused adose-dependenf inhibition of DGLA conversion to AA with a maximum of 50%inhibition at 50 μM. This study illustrates the effectiveness of addinga Δ⁵ desaturase inhibitor in conjunction with GLA in order to reduceserum arachidonic acid.

EXAMPLE 9 Determination of the Accumulation of ω-3 AA from StearidonicAcid Treatment of Neutrophils Affects the Capacity of Cells to ReleaseAA and Synthesize Eicosanoids

In additional work, the inventor has also demonstrated that humanneutrophils rapidly take up stearidonic acid (18:4, n-3) and convert itto ω-3 AA. ω-3 AA is a 20 carbon fatty acid that is a close structuralanalog of AA (n-6). Thus, ω-3 AA may also serve as a competitiveantagonist for AA (n-6) during AA metabolism. The following examplesprovide details of procedures used to investigate this strategy.

It is not known whether ω-3 AA-containing phospholipids will influencethe capacity of PLA₂ isotypes to release AA (n-6) in stimulatedneutrophils or whether ω-3 AA will effect enzymes distal tophospholipase A₂ such as 5-lipoxygenase or cyclooxygenase I and II.These issues are readily explored by ‘loading’ human neutrophils invitro with ω-3 AA; and then activating the cells. The capacity of thecells to release AA, stearidonic acid, and ω-3 AA, as well as produceeicosanoids, is examined.

Isolated neutrophils (20 million/40 ml of media) or eosinophils (10million/40 ml of media) are maintained in culture with RPMI, 2% insulintransferrin, 1% FBS and various concentrations of stearidonic acid(quantities ranging from 0 to 200 nmol). After 24 h, these cells arewashed (2×) with Hanks Balanced Salt Solution containing 0.25 mg/mlalbumin and then resuspended at a concentration of 10 million/ml. Cellsthen are stimulated with ionophore A23187 (1 μM) and maintained at 37°C. for an additional 5 min. For a more physiologic stimulus, neutrophilsare incubated in 10% autologous plasma containing 1 μg/ml LPS for 30min. Eosinophils are stimulated with PAF (1 μM). Cells are then washedand incubated with or without FMLP (1 μM). Reactions are terminated withmethanol/chloroform (2:1, v/v) or methanol, for fatty acid release orleukotriene analysis, respectively.

To determine the quantity of fatty acids released from glycerolipidsduring cell activation, octadeuterated AA and trideuterated stearic acidare added as internal standards to the terminated reaction mixture andlipids are extracted by the method of Bligh and Dyer, 1959. Fatty acidsin samples are then analyzed by NICI GC/MS. Quantities of leukotrienesare determined following reverse phase HPLC separation as describedabove. Quantities of prostaglandins are determined by NICI GC/MS. Fromthese studies, it can be determined: 1) Whether the presence of ω-3 AAor stearidonic acid-containing phospholipid in cellular membranes ofneutrophils and eosinophils influence the capacity of neutrophilphospholipase A₂(s) to mobilize AA (n-6); 2) Whether ω-3 AA orstearidonic acid is released from membrane glycerolipids during cellactivation; and 3) Whether the presence of ω-3 fatty acids affects thecapacity of neutrophils and eosinophils to synthesize leukotrienes andprostaglandins.

In addition to examining the effect of ω-3 arachidonic acid on AAmetabolism, it is also determined whether ω-3 arachidonic acid itself ismetabolized by neutrophils to eicosanoid-like products. In thesestudies, Δ²³ ¹⁸⁷-stimulated and unstimulated neutrophils are incubatedwith ω-3 arachidonic acid (from 1 to 50 μM for 10 min.). Products arethen separated by reverse phase HPLC and fractions monitored at 234 nM[HETE-like compounds] or 270 [leukotriene-like compounds]. New productsobserved with ω-3 AA and A23187 addition (not observed with eitheralone) are isolated and converted tomethoxime-pentafluorobenzyl-ester-trimethylsilyl ether derivatives asdescribed previously. Derivatized products as carboxylate anions areanalyzed by negative ion chemical ionization GC/MS. It is possible thatsome products of ω-3 AA may not absorb at the above mentionedwavelengths. In this case, there are several HPLC-electrospray massspectrometry/mass spectrometry procedures for characterizing the doublebond positions and position of hydroxyl modifications of fatty acids.These are used to definitively identify products from ω-3 AA.

It is likely that ω-3 AA attenuates the capacity of cells to synthesizeleukotrienes. Further, neutrophil PLA₂(s) hydrolyzes ω-3 AA fromcellular glycerolipids during cell activation.

EXAMPLE 10 Effects of in vivo Supplementation with Oils Enriched inStearidonic Acid (18:4, n-3) on the Quantities and Ratios of n-6 and n-3Fatty Acid in Serum and Neutrophil Lipids and the Ex Vivo Capacity ofStimulated Neutrophils from Supplemented Volunteers to Release FattyAcids and Produce Eicosanoids

It has been demonstrated that in vitro incubation (for 24 h) ofstearidonic acid with human neutrophils leads to a dramatic increase inthe quantity of ω-3 AA in cellular glycerolipids and thus a largeincrease in the ω-3 AA/AA ratio in these complex lipids. These dataindicate that the neutrophil elongase activity can be utilized tosynthesize close structural analogs of AA from appropriate dietaryprecursors. These analogs are then postulated to affect AA metabolismvia phospholipase A₂, 5-lipoxygenase or cyclooxygenase I or II.

It is contemplated that there is an in vivo dose-dependent relationshipbetween the quantity of stearidonic acid consumed in diets and thequantities of stearidonic acid, ω-3 AA and eicosapentaenoic acid inserum lipids and ω-3 AA in neutrophil lipids. If (ω-3 AA accumulates inneutrophil lipids as predicted and it acts as a competitor with AA(n-6), then it is also likely that increasing stearidonic acid doseswill correlate with a further attenuation of leukotriene generation byneutrophils and whole blood, and a concomitant increase in ω-3 AArelease from cellular phospholipids.

Recruitment of subjects, diet preparations and monitoring dietcompliance are all performed as described above. To limit variability ofvolunteers' normal diets, four randomly assigned groups of volunteers(10 per group, 5 males and 5 females) are provided identical 25% fatdiets for two weeks before starting stearidonic acid (SDA)supplementation. Then one group of volunteers consumes 1.5 g SDA/day;another group consumes 3.0 g SDA/day and a third group consumes 6.0 gSDA/day. A separate (fourth) group of subjects consumes 3.0 g of alphalinoleic acid from Crossential GLA. Crossential GLA is a commerciallyavailable oil from Croda which contains >75% of its fatty acids as alphalinolenic acid. This oil contains no stearidonic acid. This control isnecessary to test the hypothesis that bypassing the Δ⁶ desaturase isnecessary to effectively produce analogs of AA (ω-3 AA) in inflammatorycells. All groups consume their respective supplement and identicalcontrolled 25% diets for four weeks. Fasting blood is collected beforestarting the 25% diet (before diet control) and one and seven daysbefore starting the supplementation. Subsequently, fasting blood samplesare collected every 7 days after supplementation and 2 weeks aftersupplementation has ceased.

Analysis of Fatty Acids and Eicosanoids in Neutrophils and Whole Blood

Fasting (12 h) blood samples are obtained at each of the time points (inall protocols) described above. The following fatty acid and eicosanoidmeasurements are made at each time point. Eicosanoid measurements inwhole blood and in stimulated neutrophils are performed as describedabove.

Measurements of Free AA, ω-3 AA and Stearidonic Acid in StimulatedNeutrophils

Stimulated neutrophils release AA from phospholipids utilizing PLA₂(s)reactions. It is also possible that PLA₂(s) recognizes ω-3 AA orSDA-containing phospholipids, or supplementation with SDA blocks thePLA₂-induced release of AA in neutrophils. Therefore, free AA, ω-3 AA,SDA, and eicosapentaenoic acid are measured by NICI GC/MS before andafter stimulation of neutrophils isolated from each volunteer at eachdietary time point. Neutrophils are stimulated with ionophore A23187,LPS or LPS and FMLP.

It is expected that stearidonic acid (like GLA) is both elongated and Δ⁵desaturated in serum compartments to form ω-3 AA and eicosapentaenoicacid (EPA), respectively. It is also contemplated that only ω-3 AAaccumulates in neutrophil glycerolipids, thus increasing the ω-3 AA/AAratio. Stearidonic acid containing oils are also expected to induce muchhigher quantities of ω-3 AA in neutrophil lipids than alpha linolenicacid. It is likely that the accumulation of ω-3 AA translates into areduction in the capacity of blood cells, the neutrophil in particular,to produce eicosanoids. Again, one of the major advantages ofstearidonic acid versus GLA as a supplement is that the elongation/Δ⁵desaturase product from this precursor is EPA and not AA. Consequently,even if EPA accumulates in serum components, it will not enhance AAmetabolism.

EXAMPLE 11 A Preferred Embodiment of a Dietary Fatty Acid Supplement

A preferred composition would be a stabilized emulsion that can beconsumed neat or easily mixed in a drink or yogurt. The preferredcomposition of the emulsion would be:

Constituents Weight (g) Purified water 19.29 Ascorbyl palmitate 0.2Sorbic acid 0.16 Sucrose 25 Glycerin 5 Xanthan gum 0.3 Concentratedborage oil (40% GLA) 20 Concentrated marine oil (33% EPA) 15 Flavor(orange/peach) 15 Colorant (orange) 0.05 Total 100

The composition is preferably packaged in an oxygen-free environment insingle daily dosage packages made of oxygen impermeable materials suchas foil-lined pouches. The recommended daily dosage of 20 grams per daywould deliver about 1.5 grams of gammalinolenic acid and about 1.0 gramof eicosapentaenoic acid per day. The formulations preferably containnatural anti-oxidants, natural fruit flavors and natural coloringagents. The stabilized emulsion also may contain a natural sweetener andnatural preservative.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1. A method of treating asthma in a mammalian subject in need of such treatment by administering to said subject an effective amount of a composition in unit dosage form for delivery of a daily dose of said composition, said composition consisting essentially of: (i) an effective amount of γ-linolenic acid (GLA) for increasing dihomogammalinolenic acid (DGLA) levels in the inflammatory cells of said mammalian subject, thereby inhibiting the metabolism of arachidonic acid; (ii) an effective amount of a Δ⁵ desaturase inhibitor for inhibiting accumulation of arachidonic acid in the serum of said mammalian subject; and, optionally, (iii) an effective amount of a competitive inhibitor of arachidonic acid metabolism, wherein the Δ⁵ desaturase inhibitor and the competitive inhibitor are each stearidonic acid (SDA).
 2. The method of claim 1, wherein the mammalian subject is a human subject.
 3. The method of claim 1, wherein said GLA is present in an amount from about 1 gram to about 10 grams.
 4. The method of claim 3, wherein said GLA is present in an amount from about 1.5 grams to about 3 grams.
 5. The method of claim 1, wherein stearidonic acid is present in an amount from about 0.1 gram to about 15 grams.
 6. The method of claim 1, wherein the composition is a flavored drink.
 7. The method of claim 1, wherein the composition is a powder.
 8. The method of claim 1, wherein the composition is a tablet or capsule. 